The present invention relates to the field of molecular genetics and, in particular, to the production of recombinant Haemophilus influenzae high molecular weight proteins and nucleic acid molecules and vectors employed therein.
Encapsulated Haemophilus influenzae type b strains are a major cause of bacterial meningitis and other invasive infections in young children. However, the non-encapsulated or nontypeable H. influenzae (NTHi) are responsible for a wide range of human diseases, including otitis media, epiglottitis, pneumonia and tracheobronchitis. Vaccines based upon H. influenzae type b capsular polysaccharide conjugated to diphtheria toxoid (ref. 1. Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure), tetanus toxoid (ref. 2 and U.S. Pat. No. 4,496,538), or Neisseria meningitidis outer membrane protein (ref. 3) have been effective in reducing H. influenzae type b-induced meningitis, but not NTHi-induced disease (ref. 4).
Otitis media is the most common illness of early childhood, with 60 to 70% of all children, of less than 2 years of age, experiencing between one and three ear infections (ref. 5). Chronic otitis media is responsible for hearing, speech and cognitive impairments in children. H. influenzae infections account for about 30% of the cases of acute otitis media and about 60% of chronic otitis media. In the United States alone, treatment of otitis media costs between 1 and 2 billion dollars per year for antibiotics and surgical procedures, such as tonsillectomies, adenoidectomies and insertion of tympanostomy tubes. It is estimated that an additional $30 billion is spent per annum on adjunct therapies, such as speech therapy and special education classes. Furthermore, many of the causative organisms of otitis media are becoming resistant to antibiotic treatment. An effective prophylactic vaccine against otitis media is thus desirable.
During natural infection by NTHi, surface-exposed outer membrane proteins that stimulate an antibody response are potentially important targets for bactericidal and/or protective antibodies and, therefore, potential vaccine candidates. Barenkamp and Bodor (ref. 6) demonstrated that convalescent sera from children suffering from otitis media due to NTHi contained antibodies to high molecular weight (HMW) proteins. About 70 to 75% of NTHi strains express the HMW proteins and most of these strains contain two gene clusters termed hmw1ABC and hmw2ABC. The hmwA genes encode the structural HMWA proteins and the hmwb and hmwC genes are accessory genes responsible for the processing and secretion of the HMWA proteins (refs. 7, 8, 9; U.S. Pat. No. 5,603,938; Wo 97/36914). The HMWA proteins have been demonstrated to be adhesins mediating attachment to human epithelial cells (ref. 10) and only properly processed HMWA proteins appear to be effective adhesins (ref. 8). Immunization with a mixture of native HMW1A and HMW2A proteins resulted in protection in the chinchilla intrabulla challenge model of otitis media (ref. 11; WO 97/36914). The prototype hmw1A gene from NTHi strain 12 encodes a 160 kDa HMW1A protein that is processed by cleavage of a 35 kDa amino terminal fragment, generating the mature 125 kDa HMW1A protein. Similarly, the NTHi strain 12 hmw2A gene encodes a 155 kDa HMW2A protein that is processed by cleavage of a nearly identical 35 kDa amino terminal fragment to produce the mature 120 kDa HMW2A protein.
Plasmid pHMW1-15 (ref. 8) has a pT7-7 backbone (ref. 12) and contains the complete NTHi strain 12 hmw1ABC operon with 5xe2x80x2- and 3xe2x80x2-flanking regions. There are about 400 bp of 5xe2x80x2-flanking sequences located between the T7 promoter and the start of the hmw1A structural gene. Plasmid pHMW2-21 (ref. 10) has a pT7-7 backbone and contains the complete hmw2ABC operon with 5xe2x80x2- and 3xe2x80x2-flanking sequences. There are about 800 bp of 5xe2x80x2-flanking sequences located between the T7 promoter and the start of the hmw2A structural gene. The rHMW1A and rHMW2A proteins are produced in relatively low yield from plasmids pHMW1-15 and pHMW2-21.
The H. influenzae hmw1 ABC or hmw2 ABC genes can be genetically engineered to produce the mature recombinant HMW1A or HMW2A proteins by deleting the sequence encoding the 35 kDa leader sequence, that is normally removed by processing in H. influenzae. Since the leader sequence has been deleted, there should be no necessity for the hmw1BC or hmw2BC genes which serve to process and secrete the mature HMW1A and HMW2A structural proteins in H. influenzae (ref. 9). The yield of rHMW1A or rHMW2A protein can be significantly increased by deletion of the leader sequence and processing genes, however, the purified recombinant proteins are not protective. As set forth herein, the hmw1BC and hmw2BC genes or their protein products apparently contribute to the protective ability of rHMW1A and rHMW2A proteins. Such a requirement for otherwise redundant accessory genes, is unexpected.
The E. coli cer gene is thought to stabilize plasmids by preventing multimerization (ref. 13). For expression vectors with large inserts, the cer gene may be used to stabilize the plasmids.
The present invention is directed towards the provision of recombinant non-typeable H. influenzae high molecular weight proteins that are protective by providing certain nucleic acid molecules and vectors containing the same.
It has now been found that, in order to obtain recombinant high molecular weight (HMW) proteins of non-typeable Haemophilus which are protective, it is necessary to provide a vector containing only the segment of the A portion of the operon which encodes the mature HMW protein, i.e. lacking the segment of the A gene which encodes the leader sequence, and the B and C portions of the operon. It has also been found that the level of expression of the mature protein may be enhanced by including in the vector at least one additional segment which encodes the mature protein, the cer gene from E. coli or both. Accordingly, in one aspect of the present invention, there is provided a nucleic acid molecule comprising a promoter functional in E. coli and operatively coupled to a modified operon of a non-typeable strain of Haemophilus comprising A, B and C genes, wherein the A gene of the operon contains only a nucleic acid sequence which encodes a mature high molecular weight protein of the non-typeable strain of Haemophilus, and hence from which the portion of the A gene encoding the leader sequence is absent.
Any suitable promoter may be used to effect expression of the mature HMW protein in E. coli. However, it is preferred to use the T7 promoter.
The encoded mature high molecular weight protein may be HMW1 or HMW2 protein of the non-typeable Haemophilus strain. The non-typeable Haemophilus strain may be selected from the group consisting of strains 12, Joyc, K21, LCDC2, PMH1 and 15 of non-typeable Haemophilus influenzae. 
The present invention also provides the nucleotide sequences for the hmw1A and/or hmw2A genes of certain non-typeable strains of Haemophilus influenzae which have not been previously isolated, purified and expressed, along with the deduced amino acid sequences of the corresponding HMW1 and HMW2 proteins of the non-typeable Haemophilus strains.
Accordingly, in another aspect of the invention, there is provided an isolated and purified nucleic acid molecule encoding a high molecular weight (HMW) protein of a non-typeable strain of Haemophilus influenzae having:
(a) a DNA sequence selected from the group consisting of those shown in FIGS. 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 (SEQ ID NOS: 25, 27, 29, 32, 33, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 62, 64), or a sequence complementary thereto; or
(b) a DNA sequence encoding a high molecular weight protein having an amino acid sequence selected from the group consisting of those shown in FIGS. 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 (SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65), or a sequence complementary thereto.
The modified operon in the first aspect of the invention may include the mature protein encoding sequences (SEQ ID NOS: 27, 31, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72) or a DNA molecule encoding the mature protein having the amino acid sequences (SEQ ID NOS: 28, 32, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73) shown in such FIGS. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 29.
The nucleic acid molecule provided in accordance with the first aspect of the invention may further comprise a sequence containing at least one additional copy of the mature encoding region only of the operon of a non-typeable strain of Haemophilus, the cer gene of E. coli or both such segments.
The nucleic acid molecules provided in accordance with the first aspect of the invention may be incorporated into a vector, usually a plasmid vector, for transformation of E. coli for the purpose of expression of the mature protective high molecular protein of a non-typeable strain of Haemophilus.
Plasmid vectors for the latter purpose may have the identifying characteristics of a plasmid which is selected from the group consisting of:
DS-1046-1-1
JB-2507-7
BK-86-1-1
BK-35-4
BK-76-1-1
DS-2334-5
DS-2400-13
Details of the structures and preparation of such plasmid is provided in the Figures and Examples.
The present invention extends, in a further aspect thereof, to a strain of E. coli transformed by the vectors provided herein and expressing a protective high molecular weight protein of a non-typeable strain of Haemophilus. The present invention further includes an isolated and purified recombinant protective high molecular weight protein of a non-typeable strain of Haemophilus immunogenic segment or analog thereof producible by the transformed E. coli. 
The present invention further includes, in an additional aspect therein, a recombinant method for a production of a protective high molecular weight protein of a non-typeable strain of Haemophilus, which comprises:
transforming E. coli with a vector comprising the nucleic acid molecule provided in the first aspect of the invention,
growing E. coli to express the encoded mature high molecular weight (HMW) protein, and
isolating and purifying the expressed HMW protein.
The non-typeable strain of Haemophilus may be any of the strains referred to above and the high molecular weight protein may be the HMW1 protein or HMW2 protein, which is provided in a form free from contamination by the other protein. The purification steps may include separating the HMW A protein from the B and C protein.
The present invention, in an additional aspect thereof provides an isolated and purified protective HMW 1 protein of a strain of non-typeable Haemophilus which is free from contamination by the HMW2 protein of the same strain of non-typeable Haemophilus.
In a yet further aspect, the present invention provides an isolated and purified protective HMW2 protein of a strain of non-typeable Haemophilus which is free from contamination by the HMW 1 protein of the same strain of non-typeable Haemophilus.
The HMW1 or HMW2 protein may be from any of the non-typeable strain of Haemophilus mentioned above and may be one having SEQ ID NO: 28, 32, 37, 41, 45, 49, 53, 57, 61, 65, 69 or 73.
In accordance with another aspect of the invention, an immunogenic composition is provided which comprises at least one immunologically-active component selected from the group consisting of at least one nucleic acid molecule as provided herein, at least one recombinant HMW protein as provided herein or at least one novel HMW protein as provided herein, and a pharmaceutically acceptable carrier therefor. The at least one active component produces an immune response when administered to a host.
The immunogenic compositions provided herein may be formulated as a vaccine for in vivo administration to a host to provide protection against disease caused by H. influenzae. For such purpose, the compositions may be formulated as a microparticle, ISCOM or liposome preparation. The immunogenic composition may be provided in combination with a targeting molecule for delivery to specific cells of the immune system or to mucosal surfaces. The immunogenic compositions of the invention (including vaccines) may further comprise at least one other immunogenic or immunostimulating material and the immunostimulating material may be at least one adjuvant or at least one cytokine.
Suitable adjuvants for use in the present invention include (but are not limited to) aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, ISCOM matrix, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octadecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, ISCOPREP, DC-chol, DDBA and a lipoprotein and other adjuvants. Advantageous combination of adjuvants are described in copending U.S. patent applications Ser. No. 08/261,194 filed Jun. 16, 1994 and U.S. patent application Ser. No. 08/483,856 filed Jun. 7, 1995, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference (WO 95/34308, published Nov. 21, 1995).
In accordance with another aspect of the invention, there is provided a method for generating an immune response in a host, comprising the step of administering to a susceptible host, such as a human, an effective amount of the immunogenic composition as recited above. The immune response may be humoral or a cell-mediated immune response and may provide protection against disease caused by Haemophilus. Hosts in which protection against disease may be conferred include primates, including human.
It has been found that the nucleic acid sequences of the B and C portions of the operon encoding HMW1 and HMW2 proteins are highly conserved in nucleic acid sequence among species of non-typeable Haemophilus, enabling them to be provided on a universal plasmid vector for receipt of the nucleic acid sequence encoding the mature HMW1A or HMW2A protein from a variety of strains of non-typeable Haemophilus for the purpose of expression of the HMW1A or HMW2A from a transformed host, such as E. coli. 
Accordingly, in a yet further aspect of the invention, there is provided a plasmid vector for expression of a high molecular weight protein of a non-typeable strain of Haemophilus and comprising the T7 promoter, a cloning site and the B and C portions of the hmw operon of a non-typeable Haemophilus strain. The plasmid may also contain the E. coli cer gene. The plasmid vector may be plasmid JB-2646-1.
The present invention, in its various aspects, permits the production of protective high molecular weight proteins of non-typeable Haemophilus which are useful in providing immunogenic compositions to confer protection against disease caused by infection by non-typeable Haemophilus strains.